Abstract: Caffeine (1,3,7-trimethylxanthine) is a major active component of many foods, beverages, dietary supplements and
medications. Theophylline (1, 3-dimethylxanthine) is a drug used to dilate and stimulate the respiratory tract. The aims
of this study were: to determine the maximum velocity rate of cytochrome (CYP) 1A2-mediated caffeine
biotransformation using NADPH-fortified human liver microsomes (HLMs), to determine in vitro inhibitory potency
(IC50) and inhibition constant (Ki) of theophylline on caffeine biotransformation, and to provide a mechanistic
explanation for caffeine/theophylline interaction. To this end, caffeine was incubated with NADPH-fortified HLMs in
the presence and absence of theophylline. The KM and Vmax of caffeine 3-N-demethylation were determined without
adding any theophylline to the incubation; they were 0.66 ± 0.06 mM caffeine concentration and 106.3 ± 3.4 ng of
paraxanthine/hour/mg protein, respectively. The IC50 and Ki were determined after adding theophylline to the incubation;
they were 75.8 ± 5.2 ?M and 0.41 ± 0.03?M of theophylline concentrations, respectively. Our study also showed
theophylline probably acted as a competitive inhibitor of caffeine metabolism. In view of the popularity of caffeinated
beverages and adverse health effects of caffeine, care must be exercised when caffeine and theophylline are consumed
together.

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